The invention is related to the area of recombinant vaccines. It is particularly related to the field of chimeric antigens and virus-like particles for use in vaccines, especially combination vaccines for Hepatitis B virus (HBV) and Hepatitis C virus (HCV).
Hepatitis C virus (HCV) infects approximately 1% of the world""s population and causes serious health problems. Over 75% of acutely infected individuals eventually progress to a chronic carrier state that can result in cirrhosis, liver failure, and hepatocellular carcinoma. A very small fraction of chronically infected patients clear HCV naturally and resolve chronic hepatitis. See Alter et al. (1992) N. Engl. J. Med. 327:1899-1905; Resnick and Koff. (1993) Arch. Intem. Med. 153:1672-1677; Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N. Engl. J. Med. 332:1463-1466. Immunization against E2 glycoproteins of some flaviviruses (see e.g., Konishi et al., (1992) Virology 188: 714-720), including HCV (Ishii et al., (1998) Hepatology 28: 1117-1120), may protect against infection. However, attempts to express recombinant HCV E1 and E2 glyocoproteins have been frustrated by the fact that these proteins are not secreted from the host cell but are retained within the endoplasmic reticulum (Dubuisson et al. (1994) J. Virology 68: 6147-6160).
One approach to making vaccines for HCV and other viruses which has been attempted is to prepare chimeric antigens consisting of fusions of hepatitis B virus surface antigen (HBsAg) with a heterologous antigen, for example a portion of an HCV protein. See, e.g., Inchauspe et al. (1998) Dev. Biol. Stand. 92: 162-168; Nakano et al. (1997) J. Virol. (1997) 71: 7101-7109; and Inchauspe et al. (1997) Vaccine 15: 853-856. The use of HBsAg is attractive for the production of immunogenic compositions such as vaccines because HBsAg is highly immunogenic and is secreted from cultured cells in the form of virus-like particles (U.S. Pat. No. 5,098,704). Attempts to introduce small portions of viral proteins into HBsAg have succeeded in the production of virus-like particles (see e.g., Delpeyroux et al. (1990) J. Virology 64: 6090-6100, who inserted an 11 amino acid segment of polio virus capsid protein into HBsAg). However, in one study only two out of six fusion proteins containing HBsAg combined with different hydrophillic domains of HCV E2 were secreted into the culture medium as virus-like particles (Lee et al. (1996) J. Med. Virol. 50: 145-151), possibly because the E2 inserts were too large or hydrophilic. The insertion site of heterologous epitopes into HBsAg may be an important factor. A study which inserted an epitope of HBV nucleocapsid (HBcAg) at various positions into HBsAg found that insertion into an internal site in HBsAg resulted in a chimeric protein that was immunogenic for HBcAg, while insertion at the C-terminus was weakly immunogenic (Schodel et al. (1992) J. Virology 66: 106-114). Insertion at the N-terminus prevented surface access of the HBcAg epitope in the resultant particles and was non-immunogenic (Id.). Apparently, the molecular context in which an epitope is presented is important in determining immunogenicity, probably because of subtle alterations of protein secondary and tertiary structure. This principle was further illustrated by Eckhart et al. ((1996) J. Gen. Virol. 77: 2001-2008), who introduced a conserved, six amino acid epitope of HIV-1 gp41 protein into influenza hemagglutinin and obtained neutralizing antibodies, but could not generate neutralizing antibodies when the same epitope was inserted into HBsAg. Smaller isolated epitopes are more likely to be sensitive to such effects than larger portions of an immunogenic protein.
Currently there is no method available for expressing entire E1 or E2 glycoproteins of HCV in virus-like particles for use in immunization. Available methods limit chimeric proteins based on HBsAg to the insertion of only small isolated domains of E2, which may or may not have a native immunogenic structure. Thus, there remains a need in the art for methods and materials that can be used to express HCV antigens in an immunogenic form in virus-like particles.
It is an object of the invention to provide HBV/HCV chimeric antigens for use in immunogenic compositions. It is a further object of the invention to provide virus-like particles comprising HBV/HCV chimeric antigens and methods and materials for producing such virus-like particles. It is another object of the invention to provide HBV/HCV combination vaccines. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a virus-like particle for use as an immunogen or as a component of a vaccine. The virus-like particle comprises a first HBsAg (hepatitis B virus surface antigen) and a chimeric antigen. The chimeric antigen comprises a second HBsAg which is linked to an HCV immunogenic polypeptide. The first and the second HBsAg each comprise a substantially complete S domain.
Another embodiment of the invention provides another virus-like particle for use as an immunogen or as a component of a vaccine. The virus-like particle comprises a first HBsAg and first and second chimeric antigens. The first chimeric antigen comprises a second HBsAg which is linked to a first immunogenic polypeptide comprising an HCV E1 glycoprotein or a fragment thereof. The second chimeric antigen comprises a third HBsAg which is linked to a second immunogenic polypeptide comprising an HCV E2 glycoprotein or a fragment thereof. The first, second, and third HBsAg each comprise a substantially complete S domain.
Still another embodiment of the invention provides fusion proteins comprising a substantially complete S domain of HBsAg and a polypeptide. In one fusion protein, the polypeptide comprises (a) amino acid residues 192 to 330 of an HCV-1 polyprotein, or (b) the corresponding residues of other HCV isolates, or (c) an immunogenic sequence having at least about 80% sequence identity to (a) or (b). In another fuision protein, the polypeptide comprises (a) amino acid residues 384 to 661 of an HCV-1 polyprotein, or (b) the corresponding residues of other HCV isolates, or (c) an immunogenic sequence having at least about 80% sequence identity to (a) or (b).
Yet another embodiment of the invention provides nucleic acid molecules which encode fusion proteins comprising a substantially complete S domain of HBsAg and a polypeptide. In one fusion protein, the polypeptide comprises (a) amino acid residues 192 to 330 of an HCV-1 polyprotein, or (b) the corresponding residues of other HCV isolates, or (c) an immunogenic sequence having at least about 80% sequence identity to (a) or (b). In another fusion protein, the polypeptide comprises (a) amino acid residues 384 to 661 of an HCV-1 polyprotein, or (b) the corresponding residues of other HCV isolates, or (c) an immunogenic sequence having at least about 80% sequence identity to (a) or (b). These nucleic acid molecules are employed as components of immunogenic compositions, which are additional embodiments.
Another embodiment of the invention provides vectors comprising nucleic acid molecules which encode fusion proteins. The fusion proteins comprise a substantially complete S domain of HBsAg and a polypeptide comprising an immunogenic fragment of an HCV-1 polyprotein.
A further embodiment of the invention provides a method of producing virus-like particles. A cell is cultured in a culture medium, whereby the cell expresses. virus-like particles comprising a first HBsAg and a chimeric antigen. The chimeric antigen comprises a second HBsAg which is linked to an HCV immunogenic polypeptide. The first and the second HBsAg each comprise a substantially complete S domain. The virus-like particles are then isolated from the culture medium.
Yet another embodiment is a method of producing a cell line that expresses virus-like particles. A cell is transfected with a vector that expresses virus-like particles comprising a first HBsAg and a chimeric antigen. The chimeric antigen comprises a second HBsAg which is linked to an HCV immunogenic polypeptide. The first and the second HBsAg each comprise a substantially complete S domain. The cell is cultured to produce a cell line that expresses the virus-like particles.
Still other embodiments of the invention are cell lines that express virus-like particles. In one cell line, the virus-like particles comprise a first HBsAg and a chimeric antigen. The chimeric antigen comprises a second HBsAg which is linked to an HCV immunogenic polypeptide. The first and the second HBsAg each comprise a substantially complete S domain. In another type of cell line, the virus-like particles comprise a first HBsAg and first and second chimeric antigens. The first chimeric antigen comprises a second HBsAg which is linked to a first immunogenic polypeptide comprising an HCV E1 glycoprotein or a fragment thereof, and the second chimeric antigen comprises a third HBsAg which is linked to a second immunogenic polypeptide comprising an HCV E2 glycoprotein or a fragment thereof. The first, second, and third HBsAg each comprise a substantially complete S domain.
The invention thus provides the art with novel methods and materials for the production of HBV/HCV combination vaccines.